Thermophillic Organisms For Conversion Of Lignocellulosic Biomass To Ethanol

ABSTRACT

Mutant thermophilic organisms that consume a variety of biomass derived substrates are disclosed herein. Strains of  Thermoanaerobacterium saccharolyticum  with acetate kinase and phosphotransacetylase expression eliminated are disclosed herein. Further, strain ALK1 has been engineered by site directed homologous recombination to knockout both acetic acid and lactic acid production. Continuous culture involving a substrate concentration challenge lead to evolution of ALK1, and formation of a more robust strain designated ALK2. Both organisms produce near theoretical ethanol yields without expressing pyruvate decarboxylase

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/090,745, filed Apr. 18, 2008, which is a U.S. National PhaseApplication of PCT/US2006/042442, filed Oct. 31, 2006, which claimspriority to U.S. application Ser. No. 60/731,674, filed Oct. 31, 2005,and to U.S. application Ser. No. 60/796,380, filed May 1, 2006, each ofwhich is incorporated herein by reference.

GOVERNMENT INTERESTS

The United States Government may have certain rights in the presentinvention as research relevant to its development was funded by NationalInstitute of Standards and Technology (NIST) contract number60NANB1D0064.

BACKGROUND

1. Field of the Invention

The present invention pertains to the field of biomass processing toproduce ethanol. In particular, novel thermophilic organisms thatconsume a variety of biomass derived substrates and produce ethanol inhigh yield are disclosed, as well as processes for the production anduse of the organisms.

2. Description of the Related Art

Biomass represents an inexpensive and readily available cellulolyticsubstrate from which sugars may be produced. These sugars may be usedalone or fermented to produce alcohols and other products. Amongbioconversion products, interest in ethanol is high because it may beused as a renewable domestic fuel.

Significant research has been performed in the areas of reactor design,pretreatment protocols and separation technologies, so thatbioconversion processes are becoming economically competitive withpetroleum fuel technologies. However, it is estimated that the largestcost savings may be obtained when two or more process steps arecombined. For example, simultaneous saccharification and fermentation(SSF) and simultaneous saccharification and co-fermentation (SSCF)processes combine an enzymatic saccharification step with fermentationin a single reactor or continuous process apparatus. In addition tosavings associated with shorter reaction times and reduced capitalcosts, co-fermentation processes may also provide improved productyields because certain compounds that would otherwise accrue at levelsthat inhibit metabolysis or hydrolysis are consumed by the co-fermentingorganisms. In one such example, β-glucosidase ceases to hydrolyzecellobiose in the presence of glucose and, in turn, the build-up ofcellobiose impedes cellulose degradation. An SSCF process involvingco-fermentation of cellulose and hemicellulose hydrolysis products mayalleviate this problem by converting glucose into one or more productsthat do not inhibit the hydrolytic activity of β-glucosidase.

Consolidated bioprocessing (CBP) involves four biologically-mediatedevents: (1) enzyme production, (2) substrate hydrolysis, (3) hexosefermentation and (4) pentose fermentation. These events may be performedin a single step. This strategy requires a microorganism that utilizescellulose and hemicellulose. Development of CBP organisms couldpotentially result in very large cost reductions as compared to the moreconventional approach of producing saccharolytic enzymes in a dedicatedprocess step. CBP processes that utilize more than one organism toaccomplish the four biologically-mediated events are referred to asconsolidated bioprocessing co-culture fermentations.

Some bacteria have the ability to convert pentose sugars into hexosesugars, and to ferment the hexose sugars into a mixture of organic acidsand other products by glycolysis. The glycolytic pathway begins withconversion of a six-carbon glucose molecule into two three-carbonmolecules of pyruvate. Pyruvate may then be converted to lactate by theaction of lactate dehydrogenase (“ldh”), or to acetyl coenzyme A(“acetyl-CoA”) by the action of pyruvate dehydrogenase orpyruvate-ferredoxin oxidoreductase. Acetyl-CoA is further converted toacetate by phosphotransacetylase and acetate kinase, or reduced toethanol by acetaldehyde dehydrogenase (“AcDH”) and alcohol dehydrogenase(“adh”). Overall, the performance of ethanol-producing organisms iscompromised by production of organic products other than ethanol, andparticularly by ldh-mediated conversion of pyruvate to lactate, and byconversion of acetyl-CoA to acetate by phosphotransacetylase and acetatekinase.

Metabolic engineering of bacteria has recently resulted in the creationof a knockout of lactate dehydrogenase in the thermophilic, anaerobic,Gram-positive bacterium T. saccharolyticum. See, Desai, S. G.; Guerinot,M. L.; Lynd, L. R. “Cloning of L-lactate dehydrogenase and eliminationof lactic acid production via gene knockout in Thermoanaerobacteriumsaccharolyticum JW/SL-YS485” Appl. Microbiol. Biotechnol. 65:600-605,2004.

Although the knockout of ldh constitutes an advance in the art, it isproblematic for some uses of this organism in that this strain of T.saccharolyticum continues to make organic acid—in particular, aceticacid.

SUMMARY

The present instumentalities advance the art and overcome the problemsoutlined above by providing thermophilic, anaerobic bacteria thatconsume a variety of biomass derived substrates and produce ethanol innear theoretical yields. Methods for producing ethanol using theorganisms are also disclosed.

The instrumentalities reported herein result in the knockout of variousgenes either singly or in combination, where such genes in the nativeorganism would otherwise result in the formation of organic acids. Forexample, there may be knockouts of: (a) acetate kinase and/orphosphotransacetylase and (b) lactate dehydrogenase (ldh), acetatekinase (ack) and phosphotransacetylase (pta) in T. saccharolyticumJW/SL-YS485. Although the results reported herein are for T.saccharolyticum, the methods and materials also apply to other membersof the Thermoanaerobacter genus including Thermoanaerobacteriumthermosulfurigenes, Thermoanacrobacterium aotearoense,Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae,Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacteriumxylanolyticum. The methods and materials are useful generally in thefield of metabolically engineered, thermophilic, Gram-positive bacteria.

In one embodiment, an isolated organism, which does not express pyruvatedecarboxylase, ferments a cellulolytic substrate to produce ethanol in aconcentration that is at least 90% of a theoretical yield.

In one embodiment, a Gram-positive bacterium, that in a native statecontains at least one gene which confers upon the Gram-positivebacterium an ability to produce acetic acid as a fermentation product,is transformed to eliminate expression of the at least one gene. Thebacterium may be a Thermoanaerobacter, such as Thermoanaerobacteriumsaccharolyticum. The gene which confers upon the Gram-positive bacteriuman ability to produce acetic acid as a fermentation product may code forexpression of acetate kinase and/or phosphotransacetylase.

In another embodiment, the Gram-positive bacterium may be furthertransformed to eliminate expression of one or more genes that conferupon the Gram-positive bacterium the ability to produce lactic acid as afermentation product. For example, the gene that confers the ability toproduce lactic acid may be lactate dehydrogenase.

In one embodiment, a method for producing ethanol includes transforminga native organism to produce a Gram-positive bacterium that has beentransformed to eliminate expression of all genes that confer upon theGram-positive bacterium the ability to produce organic acids asfermentation products, to produce a transformed bacterial host, andculturing the transformed bacterial host in medium that contains asubstrate including a material selected from the group consisting ofglucose, xylose, cellobiose, sucrose, xylan, starch, and combinationsthereof under suitable conditions for a period of time sufficient toallow saccharification and fermentation of the substrate.

In one embodiment, a biologically pure culture of a microorganismdesignated ALK1 and deposited with the ATCC under Patent DepositDesignation No. PTA-7206 is described.

In one embodiment an isolated polynucleotide comprises (a) a sequence ofSEQ ID NO: 10, or (b) a sequence of SEQ ID NO: 9 and SEQ ID NO: 10, or(c) a sequence having at least about 90% sequence identity with thesequence of (a) or (b). A vector comprising the isolated polynucleotideof (a), (b), or (c) is described, as well as a host cell geneticallyengineered to express a compliment of the polynucleotide of (a), (b), or(c). In another embodiment, an isolated polynucleotide comprises asequence having at least about 95% sequence identity with the sequenceof (a) or (b).

In one embodiment, a method of producing ethanol includes culturing amutant bacterium expressing a compliment of the isolated polynucleotideof (a), (b), or (c) in medium containing a substrate selected from thegroup consisting of glucose, xylose, cellobiose, sucrose, xylan, starch,and combinations thereof under suitable conditions for a period of timesufficient to allow fermentation of the substrate to ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reactions of the glycolytic pathway.

FIG. 2 shows hydrogen production in wild-type T. saccharolyticumcompared to various knockout strains of T. saccharolyticum.

FIG. 3 shows a comparison of experimental and expected polynucleotidesequences for the Idh region of the suicide vector pSGD9 (SEQ ID NO: 9)integrated into the genome of T. saccharolyticum.

FIG. 4 shows a comparison of experimental and expected polynucleotidesequences for the pta/ack region of the suicide vector pSGD8-Erm (SEQ IDNO: 10) integrated into the genome of T. saccharolyticum.

FIGS. 5-7 show high-performance liquid chromotography (HPLC) traces of afermentation broth at various time intervals during growth of ALK1.

FIG. 8 shows xylose, organic acid and ethanol concentrations duringfermentation by strain ALK1.

FIG. 9 shows xylose, organic acid and ethanol concentrations duringfermentation by wild-type T. saccharolyticum.

FIG. 10 shows xylose, organic acid and ethanol concentrations during acontinuous culture challenge of ALK1.

FIG. 11 shows xylose, organic acid and ethanol concentrations duringfermentation by strain ALK2.

DETAILED DESCRIPTION

There will now be shown and described methods for engineering andutilizing thermophilic, anaerobic, Gram-positive bacteria in theconversion of biomass to ethanol.

As used herein, an organism is in “a native state” if it is has not beengenetically engineered or otherwise manipulated by the hand of man in amanner that intentionally alters the genetic and/or phenotypicconstitution of the organism. For example, wild-type organisms may beconsidered to be in a native state.

Complete elimination of organic acid production from a T.saccharolyticum in a native state was achieved using two site-directedDNA homologous recombination events. The mutant strain,Thermoanaerobacterium saccharolyticum JW/SL-YS485 ALK1 (“ALK1”) producesnear theoretical amounts of ethanol at low substrate feedings in batchculture with a temperature in a range of about 30-66° C. and a pH in arange of about 3.85-6.5. In one embodiment, ethanol yield is at leastabout 90% of the theoretical maximum. ALK1, and its decendents, have thepotential to contribute significant savings in the lignocellulosicbiomass to ethanol conversion due to its growth conditions, which aresubstantially optimal for cellulase activity in a simultaneoussaccharification and co-fermentation (SSCF) process. For example,optimal cellulase activity parameters include a pH between 4-5 andtemperature between 40-50° C. Additionally, it is unnecessary to adjustthe pH of the fermentation broth when knockout organisms, which lack theability to produce organic acids, are used. ALK1, and similar organisms,may also be suitable for a consolidated bioprocessing co-culturefermentation where the knockout organism would convert pentoses toethanol, and cellulose would be degraded by a cellulolytic organism suchas C. thermocellum.

Operating either an SSCF or CBP process at thermophilic temperaturesoffers several important benefits over conventional mesophilicfermentation temperatures of 30-37° C. In particular, costs for aprocess step dedicated to cellulase production are substantially reduced(e.g., 2-fold or more) for thermophilic SSCF and are eliminated for CBP.Costs associated with fermentor cooling and also heat exchange beforeand after fermentation are also expected to be reduced for boththermophilic SSCF and CBP. Finally, processes featuring thermophilicbiocatalysts may be less susceptible to microbial contamination ascompared to processes featuring conventional mesophilic biocatalysts.

In contrast to known “homoethanol-fermenting” microorganisms, such asnaturally-occurring Saccharomyces cerevisiae and Zymomonas mobilis, andrecombinant strains of Escherchia coli and Klebsiella oxytoca, thepresently disclosed organisms do not depend on conversion of pyruvate toacetaldehyde via the action of pyruvate decarboxylase (FIG. 1, 9). Infact, bacteria belonging to the Thermoanaerobacter genus do not expresspyruvate decarboxylase in the native state. From the reactions of theglycolytic pathway shown in FIG. 1, it can be observed that pyruvate maybe metabolized to acetyl-CoA, carbon dioxide, and reduced ferredoxin bythe enzyme pyruvate-ferredoxin oxidoreductase, 2. However, in order toproduce ethanol as the only fermentation product, the electrons carriedby reduced ferredoxin must all be transferred to NAD via NAD:ferredoxinoxidoreductase, 3, to form NADH. NADH is subsequently oxidized back toNAD in the course of the two-step reduction of acetyl-CoA to ethanol byacetaldehyde dehydrogenase, 7, and alcohol dehydrogenase, 8. Evidence ofthe efficient utilization of NADH may be observed in FIG. 2 as adecrease in production of H₂ by both the acetate knockout organism,which is unable to express ack and pta, and the double knockout organism(ALK1), which is unable to express ack, pta and ldh. These organismsprovide the first demonstration of stoichiometric electron transfer fromreduced ferredoxin to NAD, and subsequently to ethanol.

The above-described pathway, which produces stoichiometric ethanolyields in organisms that do not possess the ability to express PDC, isin contrast to the pathway employed in all previously-describedhomoethanol-fermenting strains. Previously-described strains utilizeendogenous pyruvate decarboxylase (PDC), or are engineered to expressexogenous PDC. Since expression of PDC is rare in the microbial world,the ability to redirect electron flow by virtue of modifications tocarbon flow has broad implications. For example, this approach could beused to produce high ethanol yields in strains other than T.saccharolyticum and/or to produce solvents other than ethanol. Inparticular, Gram-positive bacteria, such as members of theThermoanaerober genus; Clostridium thermocellum and other thermophilicand mesophilic Clostridia; thermophilic and mesophilic Bacillus species;Gram-negative bacteria, such as Escherichia coli and Klebsiella oxytoca;Fibrobacter succinogenes and other Fibrobacter species; Thermoganeopolitana and other Thermotoga species; and anaerobic fungi includingNeocallimatix and Piromyces species lack the ability to express PDC, andmay benefit from the disclosed instrumentalities.

It will be appreciated that the lignocellulosic material may be anyfeedstock that contains one or more of glucose, xylose, mannose,arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan,mannan and starch. In various embodiments, the lignocellulosic biomasscomprises wood, corn stover, sawdust, bark, leaves, agricultural andforestry residues, grasses such as switchgrass, ruminant digestionproducts, municipal wastes, paper mill effluent, newspaper, cardboard,or combinations thereof.

EXAMPLE 1 Production of the ALK1 Strain Materials and Methods

Thermoanaerobacterium saccharolyticum strain JW/SL-YS485 (DSM 8691) is athermophilic, anaerobic bacteria isolated from the West Thumb Basin inYellowstone National Park, Wyoming (Lui, S. Y; Gherardini, F. C.;Matuschek, M.; Bahl, H.; Wiegel, J. “Cloning, sequencing, and expressionof the gene encoding a large S-layer-associated endoxylanase fromThermoanaerobacterium sp strain JW/SL-YS485 in Escherichia coli” J.Bacteriol. 178:1539-1547, 1996; Mai, V.; Wiegel, J. “Advances indevelopment of a genetic system for Thermoanaerobacterium spp:Expression of genes encoding hydrolytic enzymes, development of a secondshuttle vector, and integration of genes into the chromosome” Appl.Environ. Microbiol. 66:4817-4821, 2000). It grows in a temperature rangeof 30-66° C. and in a pH range of 3.85-6.5. It consumes a variety ofbiomass derived substrates including the monosaccharides glucose andxylose, the disaccharides cellobiose and sucrose, and thepolysaccharides xylan and starch, but not cellulose. The organismproduces ethanol as well as the organic acids lactic acid and aceticacid as primary fermentation products.

Cloning and Sequencing

The lactate dehydrogenase (L-ldh), phosphotransacetylase (pta), andacetate kinase (ack) genes were identified and sequenced using standardtechniques, as reported previously for L-ldh (Desai, 2004). Degenerateprimers were made using the CODE-HOP algorithm (Rose, T.; Schultz, E.;Henikoff, J.; Pietrokovski, S.; McCallum, C.; Henikoff, S.“Consensus-degenerate hybrid oligonucleotide primers for amplificationof distantly-related sequences” Nucleic Acids Research, 26(7):1628-1635,1 April 1998) and PCR reactions were performed to obtain the DNAsequence between conserved regions. The gene fragments outside of theconserved regions were sequenced directly from genomic DNA usingThermoFidelase (Fidelity Systems, Gaithersburg, Md.) enzyme with BigDyeTerminator kit v3.1 (ABI, Foster City Calif.).

Construction of Suicide Vectors

Acetate Kinase and Phosphotransacetylase Knockout Vector, pSGD9

Standard cloning techniques were followed (Sambrook). The 6.2 kb suicidevector pSGD9 was based on pBLUESCRIPT II SK (+) (Stratagene) using adesign approach similar to that reported earlier (Desai, 2004; Mai,2000). Gene fragments of the pta/ack sequence, pta-up (˜1.2 kb) andack-down (˜0.6 kb), were amplified from genomic DNA using primer pairsSEQ ID NOS: 1-2, and SEQ ID NOS: 3-4. PCR amplification was performedwith pfu DNA polymerase and the fragments were extracted from a 1%electrophoresis gel. Fragments pta-up and ack-down were then A-tailedwith Taq polymerase and cloned into TOPO pCR2.1 (Invitrogen, Carlsbad,Calif.). A 1.5 kb fragment containing the kanamycin marker was obtainedfrom a PstI/XbaI digest of pIKM1 and subcloned into pBLUESCRIPT II SK(+). TOPO containing pta-up was digested with XhoI/BsiHKAI and subclonedinto XhoI/PstI digested pBLUESCRIPT II SK (+), upstream of thepreviously subcloned kanamycin marker. TOPO containing ack-down wasdigested with XbaI/SphI and subcloned into pUC19 (Invitrogen).XbaI/AflIII fragment containing ack-down was digested and subcloneddownstream of the kanamycin marker to obtain the final construct pSGD9.

Lactate Dehydrogenase Knockout Vector with Erythromycin Resistance,pSGD8-Erm

The 5.5 kb suicide vector pSGD8-Erm was based on the plasmid pSGD8 asproduced by Desai et al. 2004. In place of the aph kanamycin antibioticmarker, a fusion gene based on the aph promoter from the plasmid pIKM1and the adenine methylase gene conferring erythromycin resistance fromthe plasmid pCTC1 (Klapatch, T. R.; Guerinot, M. L.; Lynd, L. R.“Electrotransformation of Clostridium thermosaccharolyticum” J. Ind.Microbiol. 16(6):342-7, June 1996) were used for selection. PCR genefragments were created using pfu polymerase (Stategene) and the primersSEQ ID NOS: 5-6 for the aph promoter and SEQ ID NOS: 7-8 for the adeninemethylase open reading frame. Fragments were digested with XbaI/BamHl(aph fragment) and BamHI/EcoRI (adenine methylase) and ligated into themultiple cloning site of pIKM1. This fusion gene was then excised withBseRI/EcoRI and ligated into similarly digested pSGD8.

Transformation of T. saccharolyticum

Transformation of T. saccharolyticum was performed interchangeably withtwo methods, the first as previously described (Mai, V.; Lorenz, W.;Weigel, J. “Transformation of Thermoanaerobacterium sp. strainJW/SL-YS485 with plasmid PIKM1 conferring kanamycin resistance” FEMSMicrobiol. Lett. 148:163-167, 1997) and the second with severalmodifications following cell harvest and based on the method developedfor Clostridium thermocellum (Tyurin, M. V.; Desai, S. G.; Lynd, L. R.“Electrotransformation of Clostridium thermocellum” Appl. Environ.Microbiol. 70(2):883-890, 2004). Cells were grown overnight usingpre-reduced medium DSMZ 122 in sterile disposable culture tubes insidean anaerobic chamber in an incubator maintained at 55° C. Thereafter,cells were sub-cultured with 4 μg/ml isonicotonic acid hydrazide(isoniacin), a cell wall weakening agent (Hermans, J.; Boschloo, J. G.;de Bont, J. A. M. “Transformation of M. aurum by electroporation: theuse of glycine, lysozyme and isonicotinic acid hydrazide in enhancingtransformation efficiency” FEMS Microbiol. Lett. 72, 221-224, 1990),added to the medium after the initial lag phase. Exponential phase cellswere harvested and washed with pre-reduced cold sterile 200 mMcellobiose solution, and resuspended in the same solution and kept onice. Extreme care was taken following the harvesting of cells to keepthem cold (approximately 4° C.) at all times including the time duringcentrifugation.

Samples composed of 90 μl of the cell suspension and 2 to 6 μl of pSGD9or pSGD8-Erm (1 to 3 μg) added just before pulse application, wereplaced into sterile 2 ml polypropylene microcentrifuge disposable tubesthat served as electrotransformation cuvettes. A square-wave with pulselength set at 10 ms was applied using a custom-built pulsegenerator/titanium electrode system. A voltage threshold correspondingto the formation of electropores in a cell sample was evaluated as anon-linear current change when pulse voltage was linearly increased in200V increments. A particular voltage that provided the best ratio oftransformation yield versus cell viability rate at a given DNAconcentration was used, which in this particular case was 25 kV/cm.Pulsed cells were initially diluted with 500 μl DSM 122 medium, held onice for 10 minutes and then recovered at 55° C. for 4-6 hrs. Followingrecovery, cells transformed with pSGD9 were mixed with 2% agar mediumcontaining kanamycin at 75 μg/ml and poured onto petri plates andincubated in anaerobic jars for 4 days. Cells transformed with pSGD8-Ermwere allowed to recover at 48° C. for 4-6 hrs and were either plated in2% agar medium at pH 6.0 containing erythromycin at 5 μg/ml or similarliquid media and incubated in anaerobic jars at 48° C. for 6 days.Either of the transformed cell lines may be used without furthermanipulation. However, an organism where elimination of expression ofall genes that confer the ability to produce organic acids was obtainedby performing a second (sequential) transformation. The secondtransformation was carried out as described above with the primarytransformant substituted for the non- transformed cell suspension. Thesecondary transform ant, ALK1, was grown on medium containing bothkanamycin and erythromycin.

Sequencing of Knockout Regions

Sequencing of the site directed knockout regions was done by PCR fromgenomic DNA using Taq polymerase (New England Biolabs) and primersoutside the regions of homologous overlap between the genome and thesuicide vectors. Primers inside the PCR products were used forsequencing with the BigDye Terminator kit v3.1 (ABI, Foster City,Calif.). Regions were arranged using the CAP3 software program (Huang,X. “An improved sequence assembly program” Genomics 33:21-31, 1996) andcompared to the expected DNA sequence using the CLUSTALW algorithm(Higgins, D. G.; Bleasby, A. J.; Fuchs, R. “CLUSTAL V: improved softwarefor multiple sequence alignment” Computer Applications in theBiosciences (CABIOS), 8(2):189-191, 1992). A high degree of homology(percent identity) existed between the experimentally compiled sequenceand the expected sequence based on the known wild-type and suicidevector sequences (FIGS. 3 and 4).

“Identity” refers to a comparison between pairs of nucleic acid or aminoacid molecules. Methods for determining sequence identity are known.See, for example, computer programs commonly employed for this purpose,such as the Gap program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, Madison,Wis.), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl.Math. 2:482-489.

Verification of Mutant Strain

Genomic DNA from the mutant strain Thermoanaerobacterium saccharolyticumJW/SL-YS485 ALK1 (“ALK1”) showed the expected site-directed homologousrecombination in the L-ldh and pta/ack loci through DNA sequencing. Bothintegration events were double integrations, which is a more geneticallystable genotype.

EXAMPLE 2 Comparative Data Showing Production of Ethanol by ALK1 andWild-Type T. Saccharolyticum

T. saccharolyticum was grown in partially defined MTC media containing2.5 g/L Yeast Extract (Zhang, Y.; Lynd, L. R. “Quantification of celland cellulase mass concentrations during anaerobic cellulosefermentation: development of an enzyme-linked immunosorbent assay-basedmethod with application to Clostridium thermocellum batch cultures”Anal. Chem. 75:219-222, 2003). Glucose, xylose, acetate, lactate andethanol were analyzed by HPLC on an Aminex 87H column (BioRadLaboratories, Hercules, Calif.) at 55° C. The mobile phase consisted of5 mM sulfuric acid at a flow rate of 0.7 ml/min. Detection was viarefractive index using a Waters 410 refractometer (Milford, Mass.). Theminimum detection level for acetate was 1.0 mM. A standard tracecontaining 5 g/L xylose, 5 g/L lactic acid, 5 g/L acetic acid and 5 g/Lethanol is shown in FIG. 5.

Strain ALK1 produced only ethanol with up to 17 g/L xylose, or with 5g/L xylose and 5 g/L glucose, with no organic acids or other productsdetected by HPLC. FIG. 6 shows the ALK1 strain fermentation at time 0hours and FIG. 7 shows the same fermentation at 72 hours. Time coursefermentation plots of strain ALK1 and wild-type on xylose media bufferedwith 8 g/L MES at an initial pH of 6.0, 55° C. and 100 rpm show thatstrain ALK1 is able to convert over 99% of xylose to ethanol (FIG. 8),while the wild-type under similar conditions becomes pH limited due toorganic acid production and is unable to consume all the xylose present(FIG. 9). The wild-type organism yielded 0.15 mM ethanol, while ALK1yielded 0.46 mM ethanol.

EXAMPLE 3 Evolution of ALK1

As shown in FIG. 10, a continuous culture in which feed substrateconcentration was increased over time was utilized to challenge ALK1.FIG. 10 shows xylose, xylulose and ethanol concentrations during thecontinuous culture. After more than 1000 hours of exposure to thisstress-evolution cycle, an improved strain, ALK2, was isolated from thefermentation broth. ALK2 was able to initiate growth at 50 g/L xylose inbatch culture. FIG. 11 shows xylose, organic acid, optical density (OD)and ethanol concentrations during fermentation by strain ALK2.

Deposit of ALK1

ALK1 has been deposited with the American Type Culture Collection,Manassas, Va. 20110-2209. The deposit was made on Nov. 1, 2005 andreceived Patent Deposit Designation Number PTA-7206. This deposit wasmade in compliance with the Budapest Treaty requirements that theduration of the deposit should be for thirty (30) years from the date ofdeposit or for five (5) years after the last request for the deposit atthe depository or for the enforceable life of a U.S. Patent that maturesfrom this application, whichever is longer. ALK1 will be replenishedshould it become non-viable at the depository.

The description of the specific embodiments reveals general conceptsthat others can modify and/or adapt for various applications or usesthat do not depart from the general concepts. Therefore, suchadaptations and modifications should and are intended to be comprehendedwithin the meaning and range of equivalents of the disclosedembodiments. It is to be understood that the phraseology or terminologyemployed herein is for the purpose of description and not limitation.

The foregoing examples may be suitably modified for use upon anyGram-positive bacterium, and especially members of theThermoanaerobacter genus including Thermoanaerobacteriumthermosulfurigenes, Thermoanaerobacterium aotearoense,Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae,Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacteriumxylanolyticum.

All references mentioned in this application are incorporated byreference to the same extent as though fully replicated herein.

1. An isolated organism that ferments a cellulolytic substrate toproduce ethanol in a concentration that is at least 90% of a theoreticalyield, wherein the organism does not express pyruvate decarboxylase. 2.A method for producing ethanol, said method comprising: transforming anative organism to produce the isolated organism of claim 1 to provide atransformed host; and culturing the transformed host in medium thatcontains a substrate including a material selected from the groupconsisting of glucose, xylose, mannose, arabinose, galactose, fructose,cellobiose, sucrose, maltose, xylan, mannan, starch, and combinationsthereof under suitable conditions for a period of time sufficient toallow saccharification and fermentation of the substrate.
 3. Atransformed organism comprising, a Gram-positive bacterium that in anative state contains at least one gene which confers upon theGram-positive bacterium an ability to produce acetic acid as afermentation product, the Gram-positive bacterium being transformed toeliminate expression of the at least one gene.
 4. The Gram-positivebacterium according to claim 3, wherein the Gram-positive bacterium is amember of the Thermoanaerobacter genus.
 5. The Gram-positive bacteriumaccording to claim 3, wherein the Gram-positive bacterium is aThermoanaerobacterium saccharolyticum.
 6. The Gram-positive bacteriumaccording to claim 3, wherein the at least one gene codes for expressionof acetate kinase.
 7. The Gram-positive bacterium according to claim 3,wherein the at least one gene codes for the expression ofphosphotransacetylase.
 8. The Gram-positive bacterium according to claim3, wherein the at least one gene includes a plurality of genes.
 9. TheGram-positive bacterium according to claim 8, wherein the plurality ofgenes code for expression of acetate kinase and phosphotransacetylase.10. The Gram-positive bacterium according to claim 9, furthertransformed to eliminate expression of one or more genes that conferupon the Gram-positive bacterium the ability to produce lactic acid as afermentation product.
 11. The Gram-positive bacterium according to claim3, further transformed to eliminate expression of one or more genes thatconfer upon the Gram-positive bacterium the ability to produce lacticacid as a fermentation product.
 12. The Gram-positive bacteriumaccording to claim 11, wherein the at least one gene codes for theexpression of lactate dehydrogenase.
 13. A method for producing ethanol,said method comprising: transforming a native organism to produce theGram-positive bacterium of claim 11 to produce a transformed bacterialhost; and culturing the transformed bacterial host in medium thatcontains a substrate including a material selected from the groupconsisting of glucose, xylose, mannose, arabinose, galactose, fructose,cellobiose, sucrose, maltose, xylan, mannan, starch, and combinationsthereof under suitable conditions for a period of time sufficient toallow saccharification and fermentation of the substrate.
 14. The methodaccording to claim 13, wherein said bacterial host is aThermoanaerobacterium saccharolyticum.
 15. The method according to claim13, wherein the genes code for the expression of lactate dehydrogenase,acetate kinase, and phosphotransacetylase.
 16. A biologically pureculture of a microorganism designated ALK1 and deposited under PatentDeposit Designation No. PTA-7206.
 17. An isolated polynucleotidecomprising: (a) a sequence of SEQ ID NO: 10; (b) a sequence of SEQ IDNO: 9 and SEQ ID NO: 10; or (c) a sequence having at least about 90%sequence identity with the sequence of (a) or (b).
 18. Thepolynucleotide of claim 17, having about 95% sequence identity with thesequence of (a) or (b).
 19. A vector comprising the isolatedpolynucleotide of claim
 18. 20. A host cell genetically engineered toexpress a compliment of the polynucleotide of claim
 18. 21. The hostcell of claim 20, wherein the host cell is a bacterial cell.
 22. Amethod of producing ethanol comprising the step of: culturing a mutantbacterium according to claim 21 in medium containing a substrateselected from the group consisting of glucose, xylose, mannose,arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan,mannan, starch, and combinations thereof under suitable conditions for aperiod of time sufficient to allow fermentation of the substrate toethanol.
 23. The method of claim 22, wherein the mutant bacterium isThermoanaerobacterium saccharolyticum.
 24. The method of claim 23,wherein the mutant bacterium is Thermoanaerobacterium saccharolyticumALK1 (JW/SL-YS485 ALK1).
 25. A genetic construct comprising SEQ ID NO:10 operably connected to a promoter expressible in a bacterium.
 26. Arecombinant bacterium comprising the genetic construct of claim
 25. 27.The recombinant bacterium of claim 26, wherein said bacterium isThermoanaerobacterium saccharolyticum.